The field of this invention is the area of molecular biology, and in particular the DNA sequence encoding Protein Phosphatase Methylesterase-1 (PME-1, formerly called p44A), recombinant vectors, and methods for recombinant production of PME-1 demethylase and its use in identifying compositions with inhibitory activity.
Protein phosphatase 2A (PP2A) is a highly conserved serine/threonine phosphatase involved in the regulation of a wide variety of enzymes, signal transduction pathways, and cellular events [Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508; Lee, T. H., et al. (1991) Cell 64:415-423; Mayer-Jaekel, R. E. et al. (1993) Cell 72:621-633; Sontag, E. S. et al. (1993) Cell 75:887-897; Uemura, T. et al. (1993) Genes Dev. 7:429-440]. The minimal structure thought to exist in vivo consists of a heterodimer between a catalytic 36 kDa subunit termed C and a constant regulatory 63 kDa subunit termed A [Kremmer, E. et al. (1997) Mol. Cell Biol. 17:1692-1701; Usui, H. et al. (1988) J. Biol. Chem. 263:3752-3761]. This heterodimer is often further complexed with one of several additional regulatory subunits termed B, B', and B"[Cohen, P. (1989) supra]. In PP2A heterotrimers, the A subunit binds to both the catalytic C and regulatory B-type subunits [Ruediger, R. et al. (1992) J. Virol. 68:123-129; Ruediger, R. et al. (1994) Mol. Cell Biol. 12:4872-4882]. In the case of the B subunit, it has been shown that one or more of the nine C subunit carboxy terminal amino acids are essential for heterotrimer formation [Ogris, E. et al. (1997) Oncogene 15:911-917]. In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT is found in place of the B subunit in a small portion (-10%) [Ulug, et al. (1992) J. Virol. 66:1458-1467] of PP2A complexes [Pallas, D. C. et al. (1990) Cell 60:167-176]. MT/PP2A complex formation is important for MT-mediated transformation [Grussenmeyer, et al. (1987) J. Virol. 61:3902-3909; Pallas, et al. (1988) J. Virol. 62:3934-3940; Glenn, G. M. et al. (1995) J. Virol. 69:3729-3736; Campbell, K. S. et al. (1995) J. Virol. 69:3721-3728]. Unlike for B subunit, formation of PP2A heterotrimers containing MT does not require the last nine amino acid residues of the C subunit [Ogris, E. et al. (1997) supra]. The small tumor antigens (STs) of various papovaviruses also form complexes with the A and C subunits of PP2A [Pallas, D. C. et al. (1990) supra].
Consistent with the multiple important roles that PP2A plays in diverse pathways and cellular events, PP2A is highly regulated. The regulatory mechanisms include modulation by regulatory subunits or inhibitory proteins and modulation by post-translational modification of the C subunit. Subunit composition of the PP2A complex affects both catalytic activity and substrate specificity [Agostinis, P. et al. (1992) Eur. J. Biochem. 205:241-248; Favre, B. et al. (1994)J. Biol. Chem. 269:16311-16317; Scheidtmann, K. H. et al. (1991) Mol. Cell. Biol. 11:1996-2003; Sola, M. M. et al. (1991) Biochem. Biophys. Acta 1094:211-216]. In the case of B subunit, changes of up to 100 fold have been documented using cdc2 phosphorylated substrates [Agostinis, P. et al. (1992) Eur. J. Biochem. 205:241-248; Ferrigno, P. et al. (1993) Mol. Biol Cell 4:669-677; Mayer-Jaekel, R. E. et al. (1994) Journal of Cell Science 107:2609-2618; Ogris, E. et al. (1997) supra; Sola, M. M. et al. (1991) Biochem. Biophys. Acta 1094:211-216]. Two PP2A inhibitor proteins have been reported: I1PP2A (also called PHAPI) and I2PP2A (also called PHAPII or SET) [Li, M. et al. (1996) Biochemistry 34:1988-1996; Li, M. et al. (1996) Biochemistry 35: 6998-7002; Li, M. et al. (1995) J. Biol. Chem. 271:11059-11062]. These also appear to be substrate-dependent in their effects. Perusal of the NCBI GenBank and EST databases via BLAST followed by sequence comparisons using DNASTAR MegAlign software indicates the existence of three different human PHAPI isoforms encoded by different genes and the presence of multiple alternatively spliced forms of PHAPII. A Xenopus homolog of PHAPII was recently shown to interact with B-type cyclins in vitro [Kellogg, D. R. et al. (1995) J. Cell Biol. 130:661-673], but the molecular consequences of this interaction in the regulation of PP2A are not known.
The post-translational modifications of the C subunit that have been reported to modulate PP2A activity include phosphorylation and methylation. Inhibition of PP2A activity in vitro was found upon C subunit phosphorylation at either tyrosine 307 or at one or more unidentified threonine residues [Chen, J. et al. (1992) Science 257:1261-1264; Guo, H. and Damuni, Z. (1993) Proc. Nati. Acad. Sci. USA 90:2500-2504]. A similar modification may occur in vivo in response to transformation or growth stimulation [Chen, J. et al. (1994) J. Biol. Chem. 269:7957-7962]. The first indication that PP2A C subunit was methylated involved two observations. A 36 kDa SV40 small tumor antigen (ST)-associated cellular protein is a major acceptor of the methyl group from radiolabeled S-adenosyl methionine added to cell extracts [Rundell, K (1987) J. Virol. 61:1240-1243]. This ST-associated cellular protein was reported to be the PP2A C subunit [Pallas, D. C. et al. (1990) supra]. The site of methylation of the PP2A C subunit has been identified as leucine 309 [Favre, B. et al. (1994) supra; Lee, J. and Stock, J. (1993) J. Biol. Chem. 268:19192-19195; Xie, H. and Clarke, S. (1994) J. Biol. Chem. 269:1981-1984]. One study reported an approximately two-fold increase in the activity of PP2A upon methylation, adjusting for the stoichiometry of methylation [Favre, B. et al. (1994) supra]. Only phosphorylase a and the peptide substrate, phosphorylated Kemptide, were used in that study. These substrates often give similar results. Thus, it remains to be determined whether greater effects might be observed with other substrates. Based on differential antibody recognition of methylated and non-methylated C subunit, PP2A has been reported to undergo cell cycle dependent changes in methylation [Turowski, P. et al. (1995) J. Cell Biol. 129:397-410]. It is not known whether methylation of PP2A affects the subunit composition of the enzyme. Partially purified fractions of PP2A containing A/C heterodimers or A/B/C heterotrimers have both been shown to be substrates for the PP2A methyltransferase [Xie, H. and Clarke, S. (1994) supra]. There are also data which indicate that methylated C subunit can associate with SV40 ST [Rundell, K. (1987) supra].
The B subunit functions in cell cycle progression through mitosis and in cytokinesis [Healy, A. M. et al. (1991) Mol. Cell Biol. 11:5767-5780; Mayer-Jaekel, R. E. et al. (1993) supra; Uemura, T. et al. (1993) Genes Dev. 7:429-440]. In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT is found in place of the B subunit in a small portion (-10%) [Ulug, E. T. et al. supra] of PP2A complexes [Pallas, D. C. et al. (1990) supra]. MT/PP2A complex formation is known to be important for MT-mediated transformation [Campbell, K. S. et al. (1995) supra; Glenn, G. M. et al. (1995) supra; Grussenmeyer, T. et al. (1987) supra; Pallas, D. C. et al. (1988) supra], but the precise functional consequences of MT association with PP2A are still being elucidated. It was recently shown that there is a requirement for direct B/C subunit interaction to form stable heterotrimers [Ogris, E. et al. (1997) supra].
The nine carboxy-terminal amino acids of the PP2A C subunit, residues 301 to 309, include tyrosine 307, the site of phosphorylation in vitro by v-src, and two potential sites of threonine phosphorylation, residues 301 and 304. Seven of these nine residues, including threonine 304 and tyrosine 307, are found in every PP2A C subunit cloned to date. Threonine 301 is somewhat less conserved.
In order to study cellular proteins which interact with PP2A, two catalytically inactive C subunit mutants were generated and used to form stable complexes. The present invention describes the identification of one of these proteins, herein named Protein Phosphatase Methylesterase-1 (PME-1).
Due to the fact that PP2A is shown to regulate multiple cellular pathways by dephosphorylating several key proteins, there has been a long felt need in the art to understand the molecular mechanisms by which PP2A activity is modulated. The present invention describes cloning of one such modulating enzyme for human PP2A, named herein PME-1, and also shows how to produce recombinant PME-1 polypeptide, which is then used in in vitro assays to identify inhibitors for PME-1 activity.